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. Author manuscript; available in PMC: 2012 Jun 15.
Published in final edited form as: Cancer Res. 2011 May 5;71(12):4192–4204. doi: 10.1158/0008-5472.CAN-10-2442

Figure 2.

Figure 2

A. Left Panel: qRT-PCR (top panel) and immunoblot analysis (lower panel) of KMT6, KDM1, KDM5B and SirT1 in H841 cells transfected with shRNAs against respective targets or sham control sequences, demonstrating target gene knockdown. Right Panel: Immunoblot analysis demonstrating that knockdown of KMT6 and SirT1 leads to decreased levels of H3K27Me3 and H3K9Ac respectively, whereas knockdown of KDM1 and KDM5B results in increased global H3K4Me2 levels. As expected, no changes in global H3K79Me2 levels were seen in these knockdowns.

B. ChIP analysis of the NY-ESO-1, MAGE-A1 and MAGE-A3 promoters in knockdown of KMT6, KDM1, KDM5B and SirT1 in H841 cells. Knockdown of KMT6 and SirT1 was associated with decreased occupancy of these histone modifiers, with corresponding changes in H3K27Me3 and H3K9Ac, respectively; knockdown of KDM1 and KDM5B decreased occupancy of these HMTs with a corresponding increase in H3K4Me2 within the NY-ESO-1, MAGE-A1 and MAGE-A3 promoters.

C. qRT- PCR analysis demonstrating that KMT6, KDM1 and KDM5B knockdown significantly enhances DAC-mediated activation of NY-ESO-1, MAGE-A1 and MAGE-A3 in H841 cells. Effects of SirT1 knockdown were considerably less pronounced in these cells. The bars represent Mean ±SD of triplicate experiments. † P<0.05 vs DAC-shControl.

D. Pyrosequencing analysis of NY-ESO-1, MAGE-A1 and MAGE-A3 promoter methylation in H841 cells with or without knockdown of KMT6, KDM1, KDM5B or SirT1 exposed to normal media or DAC. Knockdown of these histone modifiers had variable and relatively modest effects on methylation status of these CT-X promoters. † P<0.05 vs DAC-shControl; ‡ P<0.05 vs untreated-shControl.